Method for the selective removal of acyl-functionality attached to the 2&#39; hydroxy-group of paclitaxel-related derivatives

ABSTRACT

A method for removing acyl-groups appended by an ester linkage to the 2′-hydroxyl group present in paclitaxel-related molecules comprising treatment with alcohol under non-acidic conditions. 2′,7-bis-Monochloroacetylpaclitaxel analogs are converted to their corresponding 7-monochloroacetyl derivatives by treatment with alcohol under non-acidic conditions.

BACKGROUND OF THE INVENTION

[0001] The present invention is generally directed to an efficientmethod to remove acyl-groups appended by an ester linkage to the2′-hydroxyl group present in paclitaxel-related molecules.

[0002] Paclitaxel (PAC) is an important anticancer drug. However,because of its low water solubility, the formulations needed to deliverPAC by intravenous injection are less than ideal. Thus, considerableattention has been directed toward improving the aqueous solubility ofPAC, particularly by pursuing prodrug strategies that take advantage ofthe C7 or C2′ hydroxyl groups for such incorporations. Protecting groupsthat have been used most frequently during these manipulations includesilyl,^(1,2) monochloroacetyl³ (CAC), and trichloroacetyl.⁴ Because ofits greater reactivity, several investigators have also been able tomodify the C2′ hydroxyl group directly, i.e. without protecting the C7hydroxyl functionality.⁵⁻¹⁰

[0003] Selective manipulation of the hydroxyl groups present inpaclitaxel (PAC) and in 10-deacetylpaclitaxel (DAP) to produce stableanalogs and water-soluble prodrugs, has received considerable attentionover the course of the last twenty years of PAC-related research.¹⁻¹¹Deutsch et al.³ have shown that among all of the hydroxyl groups presentin this family of compounds, the C2′ OH is the most reactive towardacylation. When PAC is treated with carbonyldiimidazole, the resultingC2′ acylated intermediate can additionally form an oxazolone derivative,an interesting side-reaction more recently encountered by de Groot etal.⁴ as well. Amino acid derivatives connected via ester linkages at theC2′ position are considerably less stable than when connected at C7 suchthat the C2′ arrangement has been extensively pursued during prodrugstrategies. Likewise, Mathew et al.⁵ has exploited the instability ofC2′ esters to produce C7-amino acid esters of PAC by partial, selectivehydrolysis of the 2′,7-bis-substituted PAC analogs at pH 7.4. Harada etal.⁶ have speculated that the instability of the C2′ amino acid estersis due to steric repulsion of the bulky groups attached to C2′ and C3′as well as to an electronic effect from these types of esters' aminogroups. The latter has also been previously implicated by zhao et al.⁷and more recently by Pendri et al.⁸ who suggested more specifically thatprotonation of the amino group could serve to assist attack of the C2′acyl functionality by external nucleophiles due to a simple inductiveeffect. Other investigators have further postulated that C2′ esters ofPAC are particularly susceptible to cleavage by various hydrolyticenzymes present in vivo.

SUMMARY OF THE INVENTION

[0004] The present invention is directed to the selective removal ofacyl-functionalities attached to the 2′-hydroxyl-group ofpaclitaxel-related derivatives by a convenient and inexpensivesolvolytic process that involves dissolving such compounds in an alcoholsuch as methanol under non-acidic conditions. The temperature and timerequired to complete the alcoholyses depend upon the nature of theacyl-function and the concentration utilized during the removalreaction. For example, non-sterically hindered acyl-functionalities arereadily removed by simply allowing dilute methanolic solutions to standat ambient temperature for 48 hours.

[0005] Because paclitaxel is an expensive starting material, thechemical manipulations for this research program were conducted on avery small scale, namely 50 mg reactions or less. This, in turn, hasallowed the inventors to follow reactions and to purify and characterizeintermediates by using HPLC. The inventors discovered that the integrityof certain of the HPLC samples appeared to undergo decomposition whenthe samples were prepared in methanol and examined overnight via an HPLCauto-sampling/injection mode. Upon further studying the decompositionprocess, it was further discovered that it applied specifically toesters which had been formed by acylating the 2′-hydroxyl-group. Thelatter represent a common chemical protection maneuver that is deployedon paclitaxel in order to chemically manipulate other portions of theoverall molecule, after which the 2′-position protecting group must thenbe cleaved in a separate step. Previous methods used to cleave such2′-position protecting groups have involved more complicated reagentsystems that do not take the reaction to completion or have involvedharsher treatments that are not as selective for the 2′-positionrelative to other portions of the paclitaxel molecule.

[0006] Several paclitaxel analogues have now been prepared whereby the2′-position protecting group has either been removed in a one-potfashion after conducting other chemistries, or has been removed in asubsequent, distinct step wherein both approaches have utilized thesimple alcoholysis reaction uncovered above.

[0007] In addition to the method's use during synthetic medicinalchemistry research, its simplicity, efficiency and environmentallyfriendly nature as a chemical conversion makes it ideally suited for usein a production chemicals scale mode. It should be appreciated thatpaclitaxel, while considered to be a wonder-drug in the fight againstcancer, is still only a “first-generation” therapeutic agent. Indeed,numerous reports from paclitaxel's use in the clinic speak toward thedesire to have improved aqueous solubility, sustained action inmultidrug resistant cancers and, like most anticancer chemotherapeuticagents, an enhanced ratio of effects in cancer versus normal cells.Research toward “second-generation” paclitaxel-like compounds has, inturn, led to compounds wherein their synthetic production oftentraverses the exact same chemical protection, further manipulation andsubsequent deprotection strategies for which the present method has thedistinct advantages as disclosed herein.

[0008] Other objects and advantages of the present invention will becomeapparent to those skilled in the art upon a review of the followingdetailed description of the preferred embodiments and the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 is a diagram showing structures of paclitaxel (PAC),10-deacetylpaclitaxel (DAP) and selected derivatives. For PAC, R═COCH₃;For DAP, R═H; For the monocloroacetyl (CAC) derivatives, a ClCH₂CO—adduct replaces one or more of the hydroxy group hydrogens located atpositions C2′, C7 and C10. For the methoxyacetyl (MAC) derivatives, aCH₃OCH₂CO— adduct replaces one or more of the hydroxy group hydrogenslocated at positions C2′, C7 and C10.

[0010]FIG. 2 is a Table 1 showing the stability of PAC and DAP CACanalogs in methanol (1 mg/ml) at ambient temperature. TR=Retention Timeon reverse phase HPLC; NMR=Nuclear Magnetic Resonance peak for the C2′H;T_(1/2)=Half-life Product=Breakdown Product (loss of 2′-acyl group).

[0011]FIG. 3 is a diagram showing a mechanistic model for themethanolysis of PAC-related 2′-esters wherein the neighboring 3′-amidenitrogen atom catalyzes the reaction by serving as a nucleophilichydrogen bond/proton acceptor.

[0012]FIG. 4 is a schematic diagram showing reaction schemes leading tocompounds 1 to 4. In both sequences, the last chemical conversion toproduce either 2 or 4 was readily accomplished by using the methanolysisreaction described herein. Compound 5 was prepared analogously to 4except that methoxyacetyl was used instead of chloroacetyl.

[0013]FIG. 5 is a Table 2 showing selected NMR proton chemical shifts indeuterated chloroform and HPLC retention times (RT) for paclitaxel andvarious derivatives.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0014] During development of an HPLC method to purify2′,7-bis-monochloroacetyl-10-deacetylpaclitaxel (2′,7-bis-CAC-DAP), theinventors discovered that the compound was unstable in methanol solutionat a concentration of 1 mg/ml. After confirming this observation andcharacterizing the breakdown product as 7-CAC-DAP the inventors furtherdiscovered that such treatments might represent a convenient, generalmethod to selectively remove ester functionalities from the 2′-positionduring synthetic manipulations of the PAC framework.¹⁴ Interestingly, wealso found that more concentrated methanolic solutions, namely 20 mg/mlfor use in preparative HPLC, did not appear to undergo solvolysis.Likewise, the use of methanol to quench unreacted acylating reagentimmediately after chloroacetylation did not appear to disturb the C2′ester, the latter reflecting a derivatized DAP concentration of 4 mg/mlin total solvent having an acidic pH. Thus, we decided to furtherinvestigate the apparent differences in the stability of several PAC andDAP CAC esters relative to concentration and acidity. The compounds weexamined are shown in FIG. 2-Table 1.

[0015] Compound abbreviations are provided in FIG. 2-Table 1 accordingto the nomenclature and numbering specified in FIG. 1. RT=Retentiontimes on an analytical HPLC (Waters) equipped with a reversed-phasecolumn (Supleco Discovery C18) using a gradient elution (60%acetonitrile from 0-2 min., linearly ramped from 60% to 75% from 2-12min., and held steady at 75% for the duration of the assay) at a flowrate of 1.2 ml/min. monitored by UV detection (225 nm). NMR (CDCl₃)values for the C2′ proton shifts are in ppm relative to TMS. T_(1/2)=Apparent half-lives. AII compounds were independently prepared andcharacterized by HPLC, NMR and elemental analyses. †Previously reportedby Rao et al. ††Previously reported by Rimoldi et al.²¹*Less than 5%breakdown after 72 hr.

[0016] A 1 mg/ml solution of 2′,7-bis-CAC-DAP in methanol was allowed tostand at ambient temperature. 20 μl samples were withdrawn at T=0, 2, 24and 48 hrs. Longer incubations were deployed for compounds showinglittle breakdown. Aliquots were assayed directly by HPLC afterestablishing that responses obtained for serial dilutions of a freshlyprepared 10 mg/ml stock solution of 2′,7-bis-CAC-DAP were linearthroughout the concentration range anticipated for the samples. A decaycurve was generated by following the decrease in compound peak areaversus time. An apparent half-life (T_(1/2)) was then calculated from asemi-log plot using Microsoft Excel. The decay curve for2′,7-bis-CAC-DAP appears to follow pseudo first-order kinetics typicalfor solvolysis reactions. The T_(1/2) value for conversion to 7-CAC-DAPis 5.5 hrs. The product growth curve matches very closely with thecompound decay curve. Similar results were obtained for all of the other2′-chloroacetylated derivatives listed in FIG. 2-Table 1 except for2′CAC-DAP whose T_(1/2) appears to be about three-times longer (18hrs.). Nevertheless, this derivative is still much more labile than thechloroacetyl esters placed at either the 7- or 10-positions (T_(1/2)values all >>72 hrs.). For example, when 7-CAC-DAP was studied for up to120 hrs., no significant breakdown to DAP was detected.

[0017] For the more concentrated experiments, a 10 mg/ml solution of2′,7-bis-CAC-DAP was allowed to stand at ambient temperature. 10 μlaliquots were withdrawn and diluted with 90 μl of acetonitrile such that20 μl samples could subjected to the HPLC analysis at the sameconcentrations used in the previous assays. As before, isolation andcrystallization of the product followed by complete characterizationconfirmed its structure as 7-CAC-DAP. It can be noted that the NMRchemical shifts for the C2′ proton among the various family members arediagnostic for loss of the 2′-CAC esters (FIG. 2-Table 1 and FIG. 5-Table 2). For the more concentrated case, however, the apparent T_(1/2)was found to be 16.2 hrs. suggesting that increased sample concentrationcan have a protective effect upon solvolysis. Likewise, when the 1 mg/mlstudies were repeated in the presence of 0.2% acetic acid, the decaycurves again suggested that there is a protective effect on hydrolysis.For the weakly acidic studies, less than 4% breakdown of2′,7-bis-CAC-DAP was observed over 48 hrs. and the calculated T_(1/2)was determined to be>1,000 hrs. Thus, the presence of monochloroaceticacid after acetylation fortuitously serves to protect the product whenmethanol is used to quench this reaction.

[0018] When identical functionalities attached to different locations onthe same molecular framework display significantly different chemicalreactivity, the involvement of neighboring groups as either catalysts orinhibitors of such reactions is likely to be operative.¹⁵ An especiallyimportant case for catalysis occurs when the participating groups canadopt orientations analogous to a favorable ring system while thereaction traverses its transition state. In 2′,7-bis-paclitaxel-relatedesters, the 2′-ester functionality resides in close proximity to the3′-amide group. The latter can provide nucleophilic catalysis via eitherits carbonyl oxygen or its nitrogen lone pair.¹⁶ Normally, the electrondensity of the nitrogen lone pair is partially displaced by itsresonance relationship with the carbonyl such that the overall system isplanar and the oxygen typically serves as the predominant nucleophile.However, in this case the adjacent phenyl ring also donates electrons tothe carbonyl such that there is a relative increase in the localizationof the lone pair on nitrogen, an increase in the nitrogen's tetrahedralcharacter, and an increase in its capacity to serve as a nucleophile.¹⁶That this type of catalysis would be expected to be very sensitive toacidification compared to having the oxygen play such a role, is in linewith the experimental observation that the overall solvolysis isdramatically attenuated in weakly acidic solutions.

[0019] As shown in FIG. 3, the 2′-ester, the 3′-amide nitrogen, and onemolecule of methanol can be placed in an arrangement wherein the sixatoms that participate in the reaction (bolded) become oriented in aspatial relationship that resembles a six-membered ring. In this model,the amide nitrogen serves as a nucleophilic hydrogen bond acceptor toeffect neighboring group catalysis of the methanolysis reaction byenhancing the nucleophilicity of the methanol oxygen. While a concertedmechanism is also possible, the resulting transesterification has beendepicted as a two-step process in order to emphasize the catalytic roleinitially played by the nitrogen lone pair.

[0020] The concept of neighboring nucleophile-assisted hydrolysis of2′-PAC esters was used by Nicolaou et al.¹¹ in their design of PACprodrugs. For example, the rate of PAC release was found to increaseacross the series HOOCCH₂XCH₂COO-2′-PAC according to theelectron-withdrawing nature of the heteroatom systems placed at X. Inthis case, however, the proposed mechanism initially involves completeremoval of the carboxylic acid proton under basic conditions such thatthe resulting anion can then effect nucleophilic attack of the estercarbonyl located, by design, to be six atoms away.

[0021] The mechanism depicted in FIG. 3 would be expected to beaccompanied by pseudo first order kinetics wherein the apparenthalf-life should be independent of substrate concentration. While thisis exactly what appears to be happening in dilute solutions, we alsoobserved that more concentrated solutions have extended half-lives. Onepossible explanation for this seeming paradox is that the PAC-relatedmaterials may be forming aggregates as their concentrations areincreased. In this regard, PAC has been observed by others to formconcentration-dependent aggregates in non-polar media such aschloroform¹⁷ and to undergo hydrophobic-driven, conformational collapseof the C13 side chain in polar media.¹⁸ While the latter wouldpresumably occur in a concentration independent manner, another way forthe lipophilic C13 side chains to avoid protic, polar solvents would beto cluster among two or more PAC molecules, a process that would bepromoted by increasing concentration. Clustering of the C13 side chains,in turn, would be expected to limit the access of methanol to the3′-amide-catalyzed, 2′-ester-reaction area. The implications of ourfindings on the pharmacological profiles and structure-activityrelationships for the PAC-related family of compounds are also importantrelative to recent models for PAC's association withmicrotubules.^(19,20) For example, Snyder et al.²⁰ has recently reporteda binding conformation in which β-tubulin's His-229 imidazole ringappears to become flanked by the phenyl rings from PAC's 3′-benzamidoand 2-benzoyl moieties. The chemical model shown herein suggests thatsuch a stacked arrangement could be further stabilized by hydrogenbonding or even proton transfer between the nucleophilic benzamidenitrogen and an imidazole N—H depending upon the latter's state ofprotonation.

[0022] As part of research to better understand the interaction ofPAC-related compounds with various components within cancercells,^(11,12) the inventors deployed CAC protection of both PAC and10-deacetylpaclitaxel (DAP) (FIG. 4). During purification of variousmixtures of the three predominate CAC derivatives obtained for DAP,namely the 2′-mono-, the 2′,7-bis- and the 2′,7,10-tri-CAC protectedmaterials, the 2′-adducts appeared to be unstable when allowed to standfor prolonged periods in methanol. Testing purified materials on a 1 mgscale confirmed this observation. Moving to a larger sample, a 40 mLsolution of 2′,7-bis-CAC-DAP was then examined at a concentration of 1mg/mL. Methanolysis of the 2′-adduct was complete after 48 hours atambient temperature. The exclusive product was isolated by evaporationof solvent, washed with cold water, and its structure characterized as7-mono-CAC-DAP according to NMR (FIG. 5-Table 2) and elemental analysis.A similar study starting with 2′,7-bis-CAC-PAC behaved exactly the sameto provide 7-mono-CAC-PAC. In this case the structural integrity for thelatter was additionally confirmed by its independent synthesis analogousto a literature procedure⁴ wherein after treatment of PAC withchloroacetic anhydride in pyridine, the reaction mixture was subjectedto 10 mM ammonia at room temperature for 1 hour prior to preparativeHPLC separation of the resulting mixture of esters.

[0023] The chloroacetylation reactions and subsequent alcoholysis can berun in a one-pot manner as long as care is taken to insure that thereaction media is no longer acidic during the second step, e.g.neutralization by addition of a tertiary amine or via a single wash withdilute sodium bicarbonate. The combination of these steps otherwiseappears to be quite general and thus the overall method provides agentle means of selectively manipulating functionality onto theC7-hydroxyl group within PAC and DAP-related systems. Thus, the methodis particularly amenable toward the production of sensitive PAC andDAP-related derivatives that intend to bear modified substituents in theC7 and/or C10 positions. Furthermore, reactions that are sluggish,including those involving more concentrated levels of substrate, can beconveniently expedited by warming the solutions at any temperature up tothe boiling point of the alcohol. Likewise, aqueous alcoholic solutions,as well as anhydrous solutions, can also be conveniently deployed as asolvent system having any percentage of water up to levels that do notinterfere with the inherent solubility of the substrate.

Experimental Section General Methods

[0024] Organic solvents and reagents were used as received from FisherScientific Co. and from Aldrich Chemical Co., respectively. Paclitaxelwas received as a gift from Yew Tree Pharmaceuticals, Ltd.Deacetylpaclitaxel was obtained by hydrolysis of paclitaxel according toa literature method ¹³ after modification to eliminate the use ofhydrogen peroxide in the presence of tetrahydrofuran. Aqueous solutionswere prepared from their respective inorganic reagents as received fromJ. T. Baker, Inc. Dry nitrogen gas was used as received from AGA Gas Co.Reactions were stirred magnetically and cooling was achieved by usingdry ice/acetone baths. Organic phases were dried over anhydrousmagnesium sulfate or potassium carbonate (J. T. Baker reagent grade) andbrought to 2 to 3° C. prior to filtering. Evaporation of solvents wasaccomplished on a Buchi rotary evaporator connected to a water aspiratorfor reduced pressure. Drying of materials within desicators was done atambient temperature and pressure/low vacuum pressure, employingDrierite/Indicating Drierite and sodium hydroxide purchased from FisherScientific. TLC was conducted by using Whatman silica bound topolyester-backed fluorescent indicator (F254) plates purchased fromFisher Scientific. Preparative HPLC was conducted on a Waters Delta Prep3000 system equipped with a Waters C18 Deltapak column. Samples wereloaded via 32% acetonitrile/water using a flow rate of 10 mL/min. and a100 mL wash. A gradient elution (32% going to 50% acetonitrile over a 50min. time period) was then applied at a flow rate of 20 mL/min. Effluentwas monitored at 225 nm and fractionated between peaks. Fractionscorresponding to pure materials were pooled and their products extractedwith dichloromethane after evaporation of the acetonitrile. AnalyticalHPLC was performed on a Waters system equipped with either a SuplecoDiscovery C18 column or a Suplecosil C18 column using a gradient elutionof 40% acetonitrile in water going to 85% over a 45 min. time period or50% acetonitrile in water going to 95% over a 45 min. time period,respectively, with both having a flow rate of 1 mL/min. monitored at 225nm. Melting points were determined on an Electrothermal digital meltingpoint apparatus and are uncorrected. Proton NMR spectra were recorded at30° C. on a Varian 400-MHz Fourier transform spectrometer in CDCl₃(Cambridge Isotope Laboratories, Inc.) using TMS as the internalstandard (δ 0.00). IR spectra were recorded at ambient temperature on aPerkin Elmer model 1600 FT-IR Spectrophotometer using 3M type 62 IRcards purchased from Aldrich Chemical Co. Elemental analyses wereperformed by Atlantic Microlab Inc.

EXAMPLE 1 Non-aqueous, Ambient Temperature Conditions

[0025] 2′,7-bis-Monochloroacetyl-10-deacetylpaclitaxel(2′,7-bis-CAC-DAP) (1). ³ 10-Deacetylpaclitaxel (DAP) (81 mg, 0.10 mmol)and dimethylaminopyridine (DMAP) (5 mg) were dissolved in 1 mL ofdimethylformamide (DMF) and a solution of monochloroacetic anhydride (43mg, 0.25 mmol) in 5 mL dichloromethane (DCM) was added. The mixture wasstirred at room temp. for 20 min. and 25 mL of DCM and 1 mL of waterwere added. The DCM solution was washed with 10 mL water, 10 mL sat.sodium bicarbonate, 10 mL water, and then dried over sodium sulfate. TheDCM was evaporated and the residue dried over NaOH to provide 85 mg. ofcrude product. Analytical HPLC indicated two peaks having an area ratioof 2 to 1. The dried residue was dissolved in 1 mL of acetonitrile andloaded on a C18 Deltapack preparative column and eluted according to thegeneral method. The effluent was fractionated and fractionscorresponding to pure products were pooled. Yield: 43 mg (0.45 mmol,45%) of 1 having 93% purity by HLPC; mp 160-163° C. (lit. ³ 166-168°C.); Analytical HPLC RT=33 min. (Discovery column); Elemental Analysiscalculated for formula C₄₉H₅₁Cl₂NO₁₅.2H₂O: C 58.80%, H 5.54%, N 1.40%.Found: C 58.52%, H 5.62%, N 1.32%. Also isolated 8 mg (0.08 mmol) of2′,7,10-tri-CAC-DAP having 98% purity by HPLC; mp 157° C. softens (lit.³ 154-155° C.); Analytical HPLC RT=35 min. (Discovery column); ElementalAnalysis Calculated for formula C₅₁H₅₂Cl₃NO₁₆.H₂O: C 57.82%, H 5.14%, N1.32%. Found: C 57.52%, H 5.11%, N 1.28%. Characteristic proton NMRchemical shifts for both products are provided in FIG. 5-Table 2.

[0026]7-Monochloroacetyl-10-deactylpaclitaxel (7-CAC-DAP) (2). 2′,7-bis-CAC-DAP (1) (30 mg, 0.031 mmol) was dissolved in 40 mL of methanol(HPLC grade) and left at ambient temperature. 20 μL samples werewithdrawn at several hour intervals and analyzed directly by HPLC. Theconversion was complete in 48 hrs. Solvent was evaporated to drynessleaving a glossy residue which crystallized upon addition of water.Yield: 25 mg (0.028 mmol) having 97% purity by HPLC; mp 163-166° C.;Analytical HPLC RT=24 min. (Discovery column); Elemental Analysiscalculated for formula C₄₇H₅₀ClNO₁₄: C 63.55%, H 5.67%, N 1.58%. Found:C 63.08%, H 5.97%, N 1.63%. Characteristic proton NMR chemical shiftsare provided in FIG. 5-Table 2.

[0027] 2′, 7-bis-Monochloroacetylpaclitaxel (2′, 7-bis-CAC-PAC) (3).Paclitaxel (43 mg, 0.05 mmol), DMAP (10 mg) and chloroacetic anhydride(40 mg, 23 mmol) were dissolved in 0.2 mL of DMF and 4 mL DCM. Themixture was allowed to react at room temperature. 20 μL samples werewithdrawn at several minute intervals and analyzed by HPLC. After about1 hour the reaction was complete. The reaction mixture was diluted with25 mL DCM and the resulting solution washed with brine. Solvent wasevaporated and the residue crystallized from methanol (2 mL) and brine(3 mL). Yield: 46 mg (0.046 mmol, 92%) having 97% purity by HPLC; mp209-211° C. (lit. ³ mp not reported); Analytical HPLC RT=39 min.(Discovery column). Characteristic proton NMR chemical shifts areprovided in FIG. 5-Table 2.

[0028]7-Monochloroacetylpaclitaxel (7-CAC-PAC) (4). 1 mg of 2′,7-bis-CAC-PAC (3) was dissolved in 1 mL of methanol and the solutionleft at ambient temperature. 20 μL samples were withdrawn at severalhour intervals and directly analyzed by HPLC. The conversion wascomplete in 48 hours. The product was identical (Table 1 HPLC and NMRdata) with 7-CAC-PAC obtained by chloroacetylation of PAC in pyridine,followed by hydrolysis using ammonia and purification by preparativeHPLC.⁴ Alternatively, 7-CAC-PAC can be obtained via a one-pot protocolstarting from PAC. Hence, after acetylation of PAC according to themethod for 3, the reaction mixture was diluted with DCM and washed withsodium bicarbonate and brine. The solution was then dried over Na₂SO₄and the solvents were removed. The 47 mg residue was dissolved in 50 mLmethanol and left at room temperature for 48 hours after which solventwas removed and the residue crystallized upon addition of water. Itshould be noted that it is important to remove all traces ofchloroacetic acid when using the one-pot process. Overall yield: 39 mg(0.48 mmol, 97%) having 99% purity by HPLC; mp 152-156° C. (darkening);Analytical HPLC RT=34 min. (Discovery column); Elemental Analysiscalculated for formula C₄₉H₅₂Cl NO₁₅: C 63.26%, H 5.63%, N 1.51%. Found:C 62.80%, H 5.93%, N. 1.41%. Characteristic proton NMR chemical shiftsare provided in FIG. 5-Table 2.

EXAMPLE 2 Non-aqueous, Elevated Temperature Conditions

[0029] 7-Methoxyacetylpaclitaxel (7-MAC-PAC) (5). 2′, 7-bis-MAC-PAC (30mg, 0.03 mmol), prepared analogously to 1 and 3, was dissolved in 60 mlof methanol and the solution stirred at 47° C. Samples (20 μl) werewithdrawn at several hour intervals and analyzed directly by HPLC.Nearly 90% conversion occurred within 7 hours. Solvent was evaporatedand the residue purified by preparative HPLC. Overall yield: 22 mg(0.024 mmol, 80%) having 99% purity by HPLC; mp 132-136° C.; AnalyticalHPLC RT=19 min. (Suplecosil column); Elemental analysis calculated forC₅₀H₅₅NO₁₆.H₂O: C 63.36%, H 6.09%, N 1.48%. Found: C 63.95%, H 6.35%, N1.35%. Characteristic proton NMR chemical shifts are provided in FIG.5-Table 2

EXAMPLE 3 Aqueous Ethanol, Ambient Temperature Conditions

[0030] 7-Monochloroacetyl-10-deacetylpaclitaxel (7-CAC-DAP) (2).2′,7-bis-CAC-DAP (0.71 mg, 0.0007 mmol) was dissolved in 0.71 ml of 80%ethanol/20% water and left at ambient temp. 20 μl samples were withdrawnat several hour intervals and analyzed directly by HPLC. Approximately20% conversion occurred in 22 hours as quantitated by analytical HPLC(substrate RT=21 min. and product RT=16 min. using the Suplecosil columnand elution protocol). The product was identified by HPLC comparisonwith an analytical sample of 7-CAC-DAP (Example 1).

EXAMPLE 4 Aqueous Ethanol, Elevated Temperature Conditions

[0031] 7-Monochloroacetyl-10-deacetylpaclitaxel (7-CAC-DAP) (2). 2′,7-bis-CAC-DAP (0.70 mg, 0.0007 mmol) was, dissolved in 0.7 ml of 80%ethanol/20% water and the solution stirred at 56° C. 20 μl samples werewithdrawn at several hour intervals and analyzed directly by HPLC.Approximately 50% conversion occurred in 24 hours as quantitated byanalytical HPLC (substrate RT=21 min. and product RT=16 min. using theSuplecosil column and elution protocol). The product was identified byHPLC comparison with an analytical sample of 7-CAC-DAP (Example 1).

[0032] The above detailed descriptions of the present invention aregiven for explanatory purposes. It will be apparent to those skilled inthe art that numerous changes and modifications can be made withoutdeparting from the scope of this invention. In particular, it isapparent that the range of paclitaxel-related derivatives spans alltypes of compounds beyond the immediate analogues of paclitaxel ordocetaxel with the only requirement being that an analogous3′-nitrogen-containing system is present and remains unobstructed sothat it can assist in the reaction according to the disclosed mechanism.Likewise, it is apparent that the range of acyl-functionality that canbe removed from the 2′-oxygen atom spans all types of compounds beyondthe simple protecting groups conveyed herein with the only requirementbeing that the immediate environment of the acyl-moiety remainsunencumbered by significant steric bulk such as would occur when theposition alpha to the carbonyl is tri-substituted with large groups. Thelatter substitution pattern is also an obvious limitation for the typesof alcohols that can be deployed during the reaction to attack theacyl-functionality. Accordingly, the whole of the foregoing descriptionis to be construed in an illustrative and not a limitative sense, thescope of the invention being defined solely by the appended claimswherein the use of the phrases “acyl-groups” and “paclitaxel-relatedmolecules,” along with the term “alcohol,” in all cases are meant toconvey the widest possible array of these compounds within the limits oftheir aforementioned constraints.

[0033] The following references are disclosed herein and are fullyincorporated herein by reference.

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We claim:
 1. A method for removing acyl-groups appended by an esterlinkage to the 2′-hydroxyl group present in paclitaxel-related moleculescomprising treatment with alcohol under non-acidic conditions.
 2. Themethod of claim 1 wherein the treatment is conducted at ambienttemperature.
 3. The method of claim 1 wherein the treatment is conductedat elevated temperature.
 4. The method of claim 2 wherein the alcoholalso contains water.
 5. The method of claim 3 wherein the alcohol alsocontains water.
 6. A method for removing acyl-groups appended by anester linkage to the 2′-hydroxyl group present in paclitaxel-relatedmolecules comprising treatment with a methanolic or ethanolic solutionunder non-acidic conditions.
 7. The method of claim 6 wherein thetreatment is conducted at ambient temperature.
 8. The method of claim 6wherein the treatment is conducted at elevated temperatures.
 9. Themethod of claim 7 wherein the alcohol also contains water.
 10. Themethod claim 8 wherein the alcohol also contains water.
 11. A method forconverting 2′,7-bis-monochloroacetylpaclitaxel,2′,7-bis-monochloracetyl-10-deacetylpaclitaxel or2′,7-bis-monochloroacetyldocetaxel to their corresponding7-monochloroacetyl derivatives by treatment with an anhydrous or aqueousalcohol system under non-acidic conditions.
 12. The method of claim 11wherein the alcohol is either methanol or ethanol and the treatment isconducted at ambient temperature.
 13. A method for converting2′,7-bis-methoxyacetylpaclitaxel,2′,7-bis-methoxyacetyl-10-deacetylpaclitaxel or2′,7-bis-methoxyacetyldocetaxel to their corresponding 7-methoxyacetylderivatives by treatment with an anhydrous or aqueous alcohol systemunder non-acidic conditions.
 14. The method of claim 13 wherein thealcohol is either methanol or ethanol and the treatment is conducted atelevated temperature.